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Glucose 6-phosphatase activity in pregnant and lactating mammary glands of the mouse.

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THE ANATOMICAL RECORD 214:383-388 (1986)
Glucose 6-Phosphatase Activity in Pregnant and
Lactating Mammary Glands of the Mouse
YOSHIHIKO SHUGYO, J U N WATANABE, SHINSUKE KANAMURA, AND
KAZUO KANA1
Department of Anatomy, Kansai Medical University, Moriguchi Osaka, 570 Japan
ABSTRACT
Glucose 6-phosphatase activity was studied in the secretory epithelial cell and other cell types composing alveoli of the mammary gland (cytochemical
study) and in the whole mammary gland (biochemical study) of pregnant and lactating mice.
The reaction product for the enzyme activity was seen in the endoplasmic reticulum and nuclear envelope in secretory epithelial cells from all animals examined
(days 7 and 14 of pregnancy, and days 0, 3, 10, and 20 of lactation. The amounts of
the reaction product appeared scarce a t day 7 of pregnancy, moderate at day 14 of
pregnancy and day 0 of lactation, and abundant at days 3 and 10 of lactation. The
reaction product, however, became generally scarce at day 20 of lactation. Biochemical activity was relatively low a t days 7 and 14 of pregnancy and days 0 and 20 of
lactation, while it was high a t days 3 and 10 of lactation.
The increased activity is probably related to functions of secretory epithelial cells
in the lactating gland.
Glucose 6-phosphatase (GGPase; EC3.1.3.9, D-glucose
6-phosphate phosphohydrolase) activity is shown to be
generally high in functionally active cells, in which the
endoplasmic reticulum is well developed, such as the
hepatocyte (Tice and Barrnett, 1962; Ericsson, 1966;
Kanamura, 1971a,b, 197513; Leskes et al., 1971) and jejunal epithelial cell (Hugon et al., 1970,1971).The secretory epithelial cell in pregnant and lactating mammary
glands is also a functionally active cell bearing welldeveloped endoplasmic reticulum and numerous secretory granules. On the other hand, glucose is present in
milk (Reineccius et al., 1970; Baldwin and Lang, 1974;
Kuhn and White, 1975). G6Pase is concerned with glucose release into tissue fluids, such as the blood (Nordlie,
1972) and epididymal fluid (Kanai et al., 1981, 1983),by
hydrolysis of glucose 6-phosphate (G6P). It is therefore
of interest to examine whether G6Pase activity is present in the secretory epithelial cell of the pregnant and
lactating mammary glands.
However, the presence of G6Pase activity in the secretory cell of the mammary gland has not been demonstrated, although there is a report about hydrolysis of
glucose 6-phosphate by homogenates of rat mammary
gland (Threadgold and Kuhn, 1979). In the present paper, we describe G6Pase activity in the secretory epithelial cell and other cell types composing alveoli of the
mammary gland (cytochemical study) and in the whole
mammary gland (biochemical study) of pregnant and
lactating mice, and discuss possible roles of the enzyme
in the mammary gland.
male for one night; the following day was designated
day 1of pregnancy. Lactating mice were used from the
time they gave birth to their litter but before nursing
(designated day 0 of lactation). Animals were examined
a t days 7 and 14 of pregnancy, and days 0 , 3 , 10, and 20
of lactation. The animals had free access to food and
water prior to the experiments.
Cytochemical methods
Left inguinal mammary glands of the animals under
Nembutal anaesthesia were perfused via the left ventricle, first with 0.9% saline for about 30 seconds, and then
with 2% glutaraldehyde containing 0.1 M sodium cacodylate (pH 7.2) for 5 minutes (3 ml/minute). The tissues
were washed briefly in 0.1 M sodium cacodylate (pH 6.7)
containing 8% sucrose a t 4°C (Kanamura, 19731, sectioned a t 40 pm by a freezing microtome, and incubated
in a reaction medium (3.7 mM G6P, 80 mM sodium
cacodylate, 3.6 mM lead nitrate, and 230 mM sucrose,
pH 6.7; Watanabe et al., 1983; Kanai et al., 1983)for 60
minutes at room temperature with a change of the medium. The sections were postfixed in 1%buffered osmium tetroxide (pH 7.4) a t 4°C for 3 hours, dehydrated,
and embedded in Spurr. Thin sections were cut with
glass knives on a n LKB ultrotome, stained with uranyl
acetate and lead citrate, and examined in a JEM 100-5
electron microscope. Observations were carried out on
alveoli of the mammary gland.
For light microscopic observations, 40-pm-thick sections incubated in the reaction medium were washed
MATERIALS AND METHODS
Female ddY mice about 3 months old were used. Pregnant mice were obtained by mating female mice with
0 1986 ALAN R. LISS, INC.
Received August 6, 1985; accepted October 30,1985.
384
Y. SHUGYO, J. WATANABE, S. KANAMURA. AND K. KANA1
with the buffer, immersed briefly in ammonium sulfide,
washed again, and mounted in glycerine jelly.
In order to ascertain whether the reaction product is
due to G6Pase activity, control experiments were carried
out (Kanamura, 1971a). The experiments consisted of
incubation of the fixed sections in the reaction medium
lacking G6P; or incubation in 0.1 M acetate buffer (pH
5.0) a t 37°C for 15 minutes before incubation in the
reaction medium; or incubation in a reaction medium
containing equal moles of 0-glycerophosphate in place of
G6P; or pre-incubation in 0.25 M sucrose containing 10
mM NaF for 15 minutes and then incubation in the
reaction medium containing equal moles of NaF.
Biochemical methods
Five animals were used per each day group of pregnancy or lactation. Mammary glands were homogenized
at 4°C in 0.25 M sucrose (2%w/v) in a Potter-Elvehjem
homogenizer for 5 minutes a t 4,000 redminute. The
homogenates were centrifuged at 3,000 redminute for
10 minutes at 4°C and assayed for G6Pase activity according to the method described by Leskes et al. (1971).
Levamisole (10 mM) was added to the reaction medium
in order to inhibit alkaline phosphatase activity. Incubation time was 30 minutes. The inorganic phosphorus
released was determined by the method of Fiske and
SubbaRow (1925). Enzyme activity was expressed as pg
phosphorus liberatediminutelmg wet tissue.
RESULTS
Cytochemical results
In secretory epithelial cells from all animals examined
(days 7 and 14 of pregnancy and days 0, 3, 10, and 20 of
lactation), the reaction product for G6Pase activity was
seen in the endoplasmic reticulum and nuclear envelope
(Figs. 1-7). The amounts of the reaction product appeared scarce at day 7 of pregnancy (Fig. 11, moderate
a t day 14 of pregnancy and day 0 of lactation (Figs. 2,
31, and abundant a t days 3 and 10 of lactation (Figs. 4,
6a). The reaction product, however, became scarce or
sometimes moderate at day 20 of lactation (Fig. 7). The
deposition of final product was occasionally found also
in lysosomes. Mitochondria, Golgi apparatus, dense-protein granules, and plasma membrane showed no reaction product.
Figs. 1-7.Cytochemical demonstration of glucose 6-phosphatase ac- product is abundant in the endoplasmic reticulum and nuclear envetivity in the cells of alveoli of mammary glands from pregnant and lope. x 13,000.
lactating mice. Sections (30 pm) cut from perfusion-fixed tissues were
Fig. 5. Secretory epithelial cell and myoepithelial cell at day 3 of
incubated in a medium modified from that of Wachstein and Meisel
lactation. Fixed sections were immersed at 37°C in 0.1 M acetate
for 60 minutes.
buffer and then incubated in the reaction medium. The reaction prodFig. 1. Secretory epithelial cells at day 7 of pregnancy. The reaction uct is absent from the endoplasmic reticulum and nuclear envelope of
product (arrowheads) is scarce in the endoplasmic reticulum and nu- secretory epithelial cell, but abundant in the plasma membrane of
myoepithelial cell. x 10,000,
clear envelope. x 15,000.
Figs. 2,3.Secretory epithelial cells at day 14 of pregnancy and day 0
of lactation, respectively. Moderate amount of the reaction product is
seen in the endoplasmic reticulum and nuclear envelope. x 10,000.
Fig. 4. Secretory epithelial cells at day 3 of lactation. The reaction
Fig. 6.Secretory epithelial cells (Fig. 6a, x 15,000)and myoepithelial
cell (Fig. 6b, ~ 8 , 0 0 0at
) day 10 of lactation. In the secretory cells, the
reaction product is abundant in the endoplasmic reticulum and nuclear
envelope. The reaction product is also seen in the endoplasmic reticulum and nuclear envelope of myoepithelial cell.
G6PASE IN MAMMARY GLAND
385
386
Y. SHUGYO, J. WATANABE, S. KANAMURA, AND K. KANA1
between day 7 of pregnancy and day 3 of lactation ( P <
.Ol), and between days 10 and 20 of lactation (P < .05)
were significant.
DISCUSSION
Fig. 7. Secretory epithelial cells at day 20 of lactation. The reaction
product is (a) scarce or (b)moderate in the endoplasmic reticulum and
nuclear envelope. x 13,000.
The reaction product for G6Pase activity was also
present in the endoplasmic reticulum and nuclear envelope in other cell types composing alveoli of pregnant
and lactating mammary glands, i.e., myoepithelial cells
(Fig. 6b), fibroblasts, and endothelial cells of capillaries.
The amount of the reaction product was moderate or
abundant in myoepithelial cells, but scarce in the other
two cell types. A scarce amount of final product was
sometimes observed also in the plasma membrane of
myoepithelial cells, fibroblasts, and endothelial cells.
Mitochondria and Golgi apparatus in these cells showed
no reaction product.
Omission of G6P from the incubation medium resulted
in a complete absence of the reaction product. Immersion of the fixed sections in 0.1 M acetate buffer (pH 5.0)
before incubation in the reaction medium or use of 0glycerophosphate in place of G6P in the reaction medium caused a loss of the reaction product except in
lysosomes and the plasma membrane. Pre-incubation of
the fixed sections in 0.25 M sucrose containing 10 mM
NaF' for 15 minutes, followed by incubation in the reaction medium containing equal moles of NaF, abolished
the total reaction, but the final product was still visible
on the plasma membrane. These results indicate that
the reaction product in the endoplasmic reticulum and
nuclear envelope is due to G6Pase activity, but the deposition of final product in the plasma membrane of myoepithelial cells, fibroblast, and endothelial cells or lysosomes in secretory epithelial cells is probably related to
nonspecific phosphatase or acid phosphatase activity.
As revealed in the present cytochemical results,
G6Pase activity in the endoplasmic reticulum and nuclear envelope of secretory epithelial cells is higher in
mammary glands of days 3 and 10 of lactation than in
pregnant and weaning glands. The biochemical activity
was relatively low at days 7 and 14 of pregnancy, and
days 0 and 20 of lactation, while it was high at days 3
and 10 of lactation. Thus, the biochemical results paralleled generally those of cytochemical experiments. The
higher G6Pase activity is probably related to functions
of secretory epithelial cells of the lactating gland.
G6Pase has a wide spectrum of hydrolytic and synthetic activities (Nordlie, 1972). However, hydrolysis of
G6P is probably the sole function of this enzyme in vivo
(Arion et al., 1972).The activity is high in hepatocytes,
proximal convoluted tubule cells of the kidney, and jejunal epithelial cells. The functional role of the enzyme
in the liver and kidney is to release glucose into blood
by hydrolyzing G6P produced via gluconeogenesis and
glycogenolysis (Krebs, 1963; Nordlie, 1972).The enzyme
in the jejunal epithelium might be concerned with the
absorption of nutrients. We recently observed relatively
high G6Pase activity in principal cells of mouse ductus
epididymidis and postulated that the role of the enzyme
is to supply glucose into the epididymal fluid for use by
spermatozoa (Kanai et al., 1981,1983). However, the role
of this enzyme in other cell types containing low or
moderate activity is unknown, although we supposed a
role of regulation of G6P concentration in the cells,
h
I
7
14
0
3
10
20
Pregnancy
(days)
Lpatm
Biochemical results
Fig. 8. G6Pase activity in pregnant and lactating mammary glands
The activity (pg phosphorus130 minuteslmg wet tissue)
of mice. Each point represents the mean of five animals and standard
was relatively low a t days 7 and 14 of pregnancy and error
is shown as a vertical line. Differences in the values between day
days 0 and 20 of lactation, while it was high at days 3 7 of pregnancy and day 3 of lactation ( P < ,011, and between days 10
and 10 of lactation (Fig. 8). Differences in the values and 20 of lactation ( P < .05) are significant.
387
GGPASE IN MAMMARY GLAND
hydrolyzing any excess (Kanamura, 1975a; Watanabe et
al.. 1983. 1986).
One role of G6Pase in secretory epithelial cells of the
lactating mammary gland may be also to regulate the
concentration of G6P in the cells. The lactating mammary gland consumes a large amount of glucose (Linzell,
1968; Lindsay, 1971). Hexokinase that synthesizes G6P
from blood glucose is shown to be high in the lactating
mammary gland (Walter and McLean, 1967). G6Pase
probably hydrolyzes G6P that is steadily produced by
hexokinase, if there is any excess.
In addition to lactose, the milk generally contains a
number of other carbohydrates. Glucose and galactose
are usually detected in low concentrations. Having taken
precautions to avoid hydrolysis of lactose, Reineccius et
al. (1970)found 0.77 mM glucose in cow’s milk. Baldwin
and Lang (1974) and Kuhn and White (1975) found 0.29
mM and 0.5-1 mM glucose in rat milk. However, we
failed to find in the literature the data of glucose in
mouse milk. Although very pronounced species differences are evident in the proportions of individual carbohydrates, it is probably impossible that certain
carbohydrates in the milk occur in one species but are
completely absent from another (Baldwin and Lang,
1974). Thus, mouse milk probably contains glucose. It is
therefore likely that one role of G6Pase in secretory
epithelial cells of the lactating mammary gland is to
release glucose into the milk by hydrolizing G6P.
Various hormones, such as estrogens, progesterone,
prolactin, glucocorticoids, growth hormone, thyroid hormones, insulin, and placental lactogen influence the
mammary development and lactation. In the mouse,
plasma concentrations of prolactin and progesterone,
after showing a peak during the first half of pregnancy,
increase toward the end of pregnancy and then fall on
the day of parturition (Murr et al., 1974; Virgo and
Bellward, 1974). Prolactin levels are high during lactation in many species (Topper and Freeman, 1980).
Plasma estradiol-170 increases close to parturition
(McCormack and Greenwald, 1974). Serum levels of corticosterone are high a t the end of pregnancy and decrease during parturition and early lactation (Gala and
Westphal, 1967). From the present results, it is not clear
which hormones are related to the changes in G6Pase
activity. Most enzyme levels in mouse mammary gland
cells are affected by prolactin, glucocorticoids, and thyroid hormones (Banerjee, 1976; Topper and Freeman,
1980).Prolactin and thyroid hormones are suggested to
regulate the rate of synthesis of the mammary enzymes
by modifying nuclear RNA synthesis (Banerjee, 1976).
Prolactin, glucocorticoids, and thyroid hormones might
relate to regulation of G6Pase synthesis.
G6Pase activity is present in a variety of cell types of
various organs, such as hepatocytes (Kanamura,
1971a,b, 1975b; Leskes et al., 1971),proximal convoluted
tubule cells of the kidney (Kanamura, 1971c), jejunal
epithelial cells (Hugon et al., 1970, 1971), pancreatic B
cells (Lazarus and Barden, 19651, follicular epithelial
cells of the thyroid gland (Rosen, 19721, cells of the
epididymis (Kanai et al., 1981, 19831, and submandibular acinar cells (Watanabe et al., 1983). In all these cell
types as well as the secretory epithelial cells in the
mammary gland, as revealed in the present results,
G6Pase activity is exclusively localized in the endoplasmic reticulum and nuclear envelope. Threadgold and
Kuhn (1979) reported that G6P hydrolysis by preparations of lactating rat mammary gland was not due to
G6Pase and could not be utilized as a microsomal
marker. However, the reaction medium used for hydrolysis of G6P contained no inhibitor for alkaline or acid
phosphatase. The authors stated as reasons for denying
G6Pase activity that G6P a s well as a wide range of
other phosphates were hydrolyzed by the mammary
gland preparation, and that the hydrolysis of G6P was
maximal a t pH 5 and 10 or above, and minimal a t pH
6.4-7. The mammary gland preparation probably includes alkaline and acid phosphatase activities, and,
therefore, such preparation hydrolyzes various phosphates. It is likely that peaks of hydrolysis at pH 5 and
10 or above are by acid and alkaline phosphatase activities, and the minimal activity at pH 6.4-7 is G6Pase
activity. As revealed in the present study, the exclusive
localization in the endoplasmic reticulum and nuclear
envelope and results of control experiments ensure the
presence of G6Pase activity in secretory epithelial cells
of the mouse mammary gland.
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